Dilated cardiomyopathy (DCM) is a disorder with genetic heterogeneity and variable phenotypes. TITIN truncating variants (TTNtvs)- nonsense, frameshift, and essential splice site, has been found to be the most common genetic factor for DCM. However, TTNtvs are also found in reference populations and it remains unclear why pathogenic TTNtvs were mainly found in exons encoding A-band domains, while TTNtvs in other exons such as those affecting the N-terminal Z-disc domain are likely benign. Moreover, patients with the same TTN mutation can exhibit highly variable disease phenotypes, presumably because of different genetic background in personal genomes. To address these two bottlenecks on TTN-based DCM, we leveraged efficient zebrafish genetics. To fill the first knowledge gap on allelic heterogeneity, we utilized genome-editing technology and generated more than 10 ttntvs affecting either Z-disc or A-band exons in zebrafish. To fill the second knowledge gap on variable phenotypes, we established a novel mutagenesis screening-based strategy and successfully identified 4 genetic modifiers for DOX-induced cardiomyopathy. We premise that some of these modifiers and related therapies for DOX-induced cardiomyopathy might also be applicable to TTN-based DCM. We propose to conduct comprehensive genetic studies of these ttntvs and candidate genetic modifiers to elucidate mechanisms and to seek therapeutic strategies for TTNtv-based DCM. In our specific Aim 1, we aim to define phenotypic traits for ttn-based DCM, and to discern toxic peptide, exon usage and cronos hypotheses for allelic heterogeneity of ttn-based DCM. In our specific Aim 2, we will elucidate novel functions of the short ttn-novex3 isoform, and test the hypothesis that N-terminal ttntvs affecting the ttn-novex3 isoform are cardiomyopathy modifiers. In our specific Aim 3, we will determine whether the 4 modifying mutants for DOX-induced cardiomyopathy also exert similar modifying effects on ttn-based DCM, and which gene can be a therapeutic target. Completion of the proposal will establish zebrafish as an efficient vertebrate model that facilitates the prognosis, diagnosis and therapeutic development for cardiomyopathies including TTN-based DCM.